JPH08500740A - Lipoprotein-related phospholipase A-2, its inhibitors and their use in diagnosis and therapy - Google Patents

Abstract

(57) Abstract: encoding enzymes _{Lp-PLA 2, Lp-PLA} 2 in purified form isolated nucleic acid molecule, used in the treatment of inhibitors of the enzyme Lp-PLA _2, and compounds that inhibit the enzyme Methods of screening compounds for identification.

Description

DETAILED DESCRIPTION OF THE INVENTION Lipoprotein-related phospholipase A ₂ The present invention relates to the use of enzyme inhibitors in therapy, in particular in the treatment of atherosclerosis. The present invention also provides for isolation and purification of the enzyme, isolated nucleic acid encoding the enzyme, recombinant host cells transformed with DNA encoding the enzyme, susceptibility of patients to atherosclerosis. It relates to the use of the enzyme in diagnosis and in the identification of compounds potentially useful in the treatment of atherosclerosis. Also, lipoprotein-related phospholipase A ₂ (Lp-PLA ₂ ) Is already known in the art as platelet activating factor acetylhydrolase (PAF acetylhydrolase). During the conversion of LDL to its oxidized form, Lp-PLA ₂ Causes hydrolysis of the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso-phosphatidylcholine and oxidatively modified fatty acids. Lp-P LA ₂ Both of these products of action are potent chemoattractants for circulating monocytes. It is believed that this enzyme, by itself, causes the accumulation of cholesterol-ester-filled cells within the arteries, causing the characteristic "fatty streak" associated with the early stages of atherosclerosis. There is. Therefore, Lp-PLA ₂ Inhibition of the enzyme (by inhibiting the formation of lysophosphatidylcholine) is expected to prevent the formation of this fatty streak and is therefore useful in the treatment of atherosclerosis. Also, Lp-PLA ₂ Have been proposed to play a direct role in LDL oxidation. This contributes to and promotes the overall oxidation process, Lp-PLA ₂ The action is due to lipid peroxide products derived from polyunsaturated fatty acids. In line with this idea, Lp-PLA ₂ The inhibitor inhibits LDL oxidation. Therefore, Lp-PLA ₂ Inhibitors include any disorder involving lipid peroxidation along with the enzyme activity, such as rheumatoid arthritis, stroke, myocardial infarction, reperfusion injury, and acute and acute as well as symptoms such as atherosclerosis and diabetes. It has general applicability to other conditions such as chronic inflammation. Therefore, in a first aspect, the present invention provides a lipoprotein-related enzyme, Lp-PLA, for use in therapy, particularly in the treatment of atherosclerosis. ₂ To provide an inhibitor of Lp-PLA ₂ Suitable compounds capable of inhibiting the enzyme are known in the art, and include, for example, the compounds represented by the following formula (I). [Wherein R is C _1-6 Alkyl conr ² ; R ² Is hydrogen or C _1-6 Alkyl; X is oxygen, sulfur or -O (CO)-; R ¹ Is C _8-20 Alkyl; Z is N (R ³ ) ₂ , ⁺ N (R ³ ) ₃ , ⁺ SR ³ , ⁺ S (R ³ ) ₂ (In each group, R ³ Are the same or different and are C _1-6 Alkyl, OR ² , C _1-4 Alkanoyl, imidazolyl or N-methylimidazolyl. Suitably R ² Is hydrogen or C _1-6 Alkyl, preferably R ² Is hydrogen, suitably X is oxygen, sulfur or -O (CO)-; preferably X is oxygen, suitably R ¹ Is C _8-20 Alkyl; preferably R ¹ Is C _16-18 Alkyl, suitably Z is N (R ³ ) ₂ , ⁺ N (R ³ ) ₃ , SR ³ , ⁺ S (R ³ ) ₂ (In each group, R ³ Are the same or different and are C _1-6 Alkyl, OR ² , C _1-4 Alkanoyl, imidazolyl or N-methylimidazolyl; preferably Z is SR ³ (Where R ³ Is methyl or OR ² , R ² Are hydrogen)] The compounds of formula (I) can be prepared by methods known to those skilled in the art, for example, Journal of Chemical Society Chemical Communication (J Chem Soc Chem Comm), 1993, 70-72; By the methods described in J. Org Chem, 1983, 48, 1197 and Chem Phys Lipids, 1984, 35, 29-37, or similar methods. It can be manufactured. When used in therapy, the compounds of formula (I) are formulated according to standard pharmaceutical practice. The compounds of formula (I) and their pharmaceutically acceptable salts which are active when administered orally can be formulated as solutions, for example syrups, suspensions or emulsions, tablets, capsules and lozenges. Liquid formulations generally include suitable liquid carrier (s) including suspending agents, preservatives, flavoring or coloring agents, such as ethanol, glycerin, non-aqueous solvents such as polyethylene glycols, oils or water. It consists of a suspension or solution of the compound or a pharmaceutically acceptable salt. The composition in the form of tablets may be prepared with any suitable pharmaceutical carrier commonly used to prepare solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose, cellulose and the like. Compositions in the form of capsules can be manufactured using conventional encapsulation methods. For example, pellets containing the active ingredient may be prepared with standard carriers and then filled into hard gelatin capsules, or with any suitable pharmaceutical carrier such as aqueous gums, celluloses, silicates or oils. Dispersions or suspensions can be prepared and then filled into soft gelatin capsules. A typical parenteral composition is a solution or suspension of the compound or a pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil such as polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil or sesame oil. It consists of a suspension. Alternatively, the solution can be lyophilized and then reconstituted with a suitable solvent just prior to administration. A typical suppository formulation may consist of a compound of formula (I) or a pharmaceutically acceptable salt thereof active when so administered, and a binder and / or a lubricant such as polymeric glycols, gelatin or It consists of cocoa butter or other low melting vegetable or synthetic waxes or fats. Preferably the composition is in unit dose form such as a tablet or capsule. Each dosage unit for oral administration preferably contains 1-250 mg (preferably 0.1-25 mg for parenteral administration) of the compound of formula (I) or a pharmaceutically acceptable thereof, calculated as the free base. It contains a salt. A daily dosage regimen for an adult patient may be, for example, an oral dose of 1 mg to 500 mg, preferably 1 mg to 250 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base, or 0 The compound may be administered at an intravenous, subcutaneous or intramuscular dose of 1 mg to 100 mg, preferably 0.1 mg to 25 mg, 1 to 4 times daily. Suitably, the compound is administered during continuous treatment periods. Enzyme, lipoprotein related Lp-PLA ₂ Has not heretofore been obtained in isolated purified form. Therefore, in another aspect, the invention provides a purified form of the lipoprotein-related enzyme Lp-PLA. ₂ I will provide a. By purified form is meant at least 80%, more preferably 90%, even more preferably 95%, and most preferably 99% pure relative to other protein contaminants. Enzyme Lp-PLA ₂ Is one or more partial peptide sequences selected from SEQ ID NO: 1, 2, 3, 4, 10 and 11, or consisting of residues 271 to 441 of SEQ ID NO: 9 or residues 1 to 441. It can be characterized by a partial peptide sequence consisting of a group. Additionally or separately, the enzyme Lp-PLA ₂ Can be characterized by its molecular weight which has been found to be 45 kDa, at least 45 kDa, 45-47 kDa, 46-47 kDa or 45-50 kDa. The present invention also provides Lp-PLA ₂ A fragment of the enzyme having activity is provided. The enzyme can be isolated and purified using the methods described below. Once isolated, the protein sequence of the enzyme can be obtained using standard techniques. In identifying the sequence, several protein fragments have been identified, each consisting of a portion of the entire sequence of the enzyme. These sequences are novel in their own right and form another aspect of the invention. The invention also provides isolated nucleic acid molecules that encode the enzyme, including mRNAs, DNAs, cDNAs and antisense analogs thereof, and biologically active and diagnostically or therapeutically useful fragments thereof. provide. In particular, the invention provides an isolated nucleic acid molecule consisting of bases 1-1361 or 38-1361, or of a sequence corresponding to bases 848-1361 of SEQ ID NO: 9. The invention also provides recombinant prokaryotic and / or eukaryotic host cells comprising a recombinant vector such as a cloning and expression plasmid useful as a reagent in the recombinant production of the enzyme, and a novel nucleic acid sequence. The invention also provides a nucleic acid probe comprising a nucleic acid molecule of sufficient length to hybridize specifically with the novel nucleic acid sequence. The present invention also provides an antisense oligonucleotide having a sequence capable of binding to the mRNA so as to avoid translation of the mRNA encoding the enzyme. The invention also provides a transformed non-human animal comprising a nucleic acid molecule encoding the enzyme. Also provided is a method of using such transformed animals for mutation and SAR (structure / activity relationship) evaluation and as a model in drug screening. Further, the invention comprises contacting the isolated enzyme with a test compound and measuring the enzyme substrate turnover rate compared to the turnover rate in the absence of the test compound. Provided are methods of screening compounds to identify those that inhibit. A "recombinant" polypeptide refers to a polypeptide produced by recombinant DNA technology, ie, produced from a cell transformed with an exogenous DNA construct encoding the desired polypeptide. A "synthetic" polypeptide is a polypeptide produced by chemical synthesis. A "replicon" is any genetic element (eg, plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, ie, has the ability to replicate under its own control. A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. "Double-stranded DNA molecule" refers to a polymeric form of deoxyribonucleotides (base adenine, guanine, thymine or cytosine) in a double helix (both relaxed and supercoiled). The term refers only to the primary and secondary structure of the molecule and is not limited to any individual tertiary forms. Thus, the term includes double-stranded DNA found among others in linear DNA molecules (eg, restriction fragments), viruses, plasmids and chromosomes. In discussing the structure of individual double-stranded DNA molecules, the sequences are described herein according to the usual rules of providing only the 5'to 3'sequence along the sense strand of DNA. It may be. A "sequence encoding" DNA for an individual protein or a nucleotide sequence "encoding for an individual protein" is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 'direction) coding sequence. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), and a protein binding domain (consensus sequence) that causes binding of RNA polymerase. Eukaryotic promoters often (but not always) contain "TATA" boxes and "CAT" boxes. A DNA "regulatory sequence" includes a promoter sequence, a ribosome binding site, a polyadenylation signal, a transcription termination sequence, an upstream regulatory domain, an enhancer, etc. that collectively prepare for the expression (ie transcription and translation) of a coding sequence in a host cell. Mean collectively. When RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence, the control sequences are "expressed" in the cell. Command. " A "host cell" is a cell that has been transformed or transfected with, or has the ability to be transformed or transfected with, an exogenous DNA sequence. A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into the chromosomal DNA that makes up the cell's genome. For example, in prokaryotes or yeast, exogenous DNA may be maintained on episomal elements such as plasmids. Regarding eukaryotic cells, stably transformed or transfected cells are those in which exogenous DNA has been integrated into the chromosome such that it is inherited by daughter cells by chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of a population of daughter cells containing exogenous DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. If at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule and contain allelic variations, two DNA or polypeptide sequences are "substantially homologous" or "substantially identical." As used herein, substantially homologous also means sequences that show identity with the identified DNA or polypeptide sequences. DNA sequences that are substantially homologous can be identified, for example, in Southern hybridization experiments under stringent conditions determined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, for example, Current Protocols in Mol. Biol. Vol. I & II, Wiley Interscience, Ed. Ausbel et al. (1992). Substantially identical protein sequences can be identified by proteolytic digestion, gel electrophoresis and microsequencing. The term “functionally equivalent” means that the amino acid sequence of the target protein is Lp-PLA. ₂ It means that it shows the same kind of enzyme activity as. A "heterologous" region of a DNA construct is an identifiable segment of DNA within or associated with another DNA molecule that is not naturally found with the rest of the molecule. The present invention relates to the enzyme Lp-PLA ₂ There is provided an isolated nucleic acid molecule encoding One means of isolating the encoding nucleic acid is to use a human genomic or cDNA library with art-recognized techniques [eg, "Current Protocols in Molecular Biology"]. , Ausubel, FM, et al., Greene Publishing Assoc. And John Wiley Interscience, New York, 1989, 1992]. Probing with a naturally or artificially designed probe, eg, using the protein fragment information disclosed herein. The enzyme of the present invention can be produced by recombinant genetic engineering technology. The isolated nucleic acid, particularly DNA, can be introduced into an expression vector by operably binding the DNA to an essential expression control region (eg, regulatory region) required for gene expression. The vector may be provided in a suitable host cell such as a prokaryotic (eg bacterial) or eukaryotic (eg yeast, insect or mammalian) cell by methods well known in the art (Au subel et al., Supra). Can be introduced to. The coding sequence for the desired protein, which has been prepared or isolated, can be cloned into a suitable vector or replicon. Large numbers of cloning vectors are known to those of skill in the art, and choosing the proper cloning vector is a matter of choice. Recombinant DNA vectors for cloning and host cells with which they can be transformed include, for example, bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli). ), PKT230 (Gram-negative bacterium), pGV1106 (Gram-negative bacterium), pLA FR1 (Gram-negative bacterium), pME290 (Non-E. Coli Gram-negative bacterium), pHV1 4 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), baculovirus (Baculovirus) insect cell line, YCp19 Saccharomyces. And so on. Generally, "DNA Cloning": Vol I & II, edited by Glover et al., IRL Press Oxford (1985) (1987) and; T. Maniatis. (Molecular Cloning, Cold Spring Harbor Laboratory) (1982). The gene is driven by a promoter, a ribosome binding site (for bacterial expression) and optionally an operator so that the DNA sequence encoding the desired protein can be transcribed into RNA in a host cell transformed with the vector containing this expression construct. (Collectively referred to herein as "control" elements). The coding sequence may or may not contain a signal peptide or leader sequence. The protein sequences of the invention can be expressed using, for example, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequences. Leader sequences can be removed by bacterial hosts in post-translational processing (see, eg, US Pat. Nos. 4,431,739; 4,425,437; 4,338,397). In addition to control sequences, it may be desirable to add regulatory sequences that allow the regulation of the expression of protein sequences associated with growth of the host cell. Regulatory sequences are known to those of skill in the art and include, for example, those that express genes that are switched on and off in response to chemical or physical stimuli, including the presence of regulatory compounds. Also, other types of regulatory elements may be present in the vector, eg, enhancer sequences. The expression vector is constructed so that the individual coding sequences are located in the vector along with appropriate regulatory sequences. Here, the placement and orientation of the coding sequence with respect to the control sequence is such that the coating sequence is transcribed under the "control" of the control sequence (ie, RNA polymerase that binds to the DNA molecule at the control sequence transcribes the coding sequence). It is like this. For this purpose, it is desirable to modify the coding sequence. For example, in some cases it may be necessary to modify the sequence so that it is attached to the control sequences in the proper orientation, ie, to maintain the reading frame. Control and other regulatory sequences may be ligated to coding sequences prior to insertion into a vector such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. Modifications of the coding sequences may also be made to alter codon usage so that it is compatible with the host cell chosen and expression is increased. In some cases, it may be desirable to add sequences that cause secretion of the polypeptide from the host organism and then cleave the secretion signal. It may also be desirable to produce mutants or analogs of the enzyme of interest. Mutants or analogs are prepared by deleting a portion of the sequence encoding the protein, inserting the sequence, and / or replacing one or more nucleotides in the sequence. Good. Techniques for modifying nucleotide sequences such as site-directed mutagenesis are well known to those of skill in the art (eg, T. Maniatis et al., Supra; DNA Cloning, VolI. And II; see above Nucleic Acid Hybridization). Several prokaryotic expression vectors are known in the art. For example, U.S. Pat. Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418. , 149; 4, 411, 994; 4,366, 246; 4,342, 832. See also British Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expression vectors are also known in the art. See, for example, U.S. Patents 4,446,235; 4,443,539; 4,430,428. See also European Patent Application Nos. 103,409; 100,561; 96,491 [pSV2 neo, which causes expression in mammalian cells using the SV40 late promoter. Of Molecular and Applied Genetics (J. Mol. Appl. Genet. 1: 327-341), or pCDNA1 (Molecular Cell Biology) that causes expression using a CMV promoter. (Mol. Cell Biol.) 7: 4125-29) -derived vector pCDNA1 neo]. These latter two vectors can be used for transient or stable (using G418 resistance) expression in mammalian cells. Also useful are insect cell expression systems (eg, Drosophila) (see, eg, PCT applications US89 / 05155 and US91 / 06838 and European Patent Application No. 88 / 304093.3). By growing a host transformed with the above expression vector under conditions in which the protein of interest is expressed, the enzyme of the present invention can be produced depending on the selected expression system and host. The protein is then isolated from the host and purified. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from cell lysates or recovered from cell membrane fractions. If the protein is localized on the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product. Protein expressing bacterial hosts such as E. coli may require isolation and regeneration from inclusion bodies. Selection of appropriate growth conditions and recovery methods are within the skill of the art. Also, the identification of this novel target for the treatment of atherosclerosis will lead to a new diagnostic method to diagnose a patient's susceptibility to the development of atherosclerotic disease. Therefore, in yet another aspect of the invention, a blood sample is isolated from a patient and the sample is isolated from Lp-PLA. ₂ A diagnostic method characterized by assaying for activity is provided. Patients susceptible to atherosclerotic disease have elevated Lp-PLA ₂ It is expected to have enzyme levels and therefore Lp-PLA ₂ Levels are indicative of a patient's susceptibility to atherosclerotic disease. Furthermore, Lp-PLA ₂ Has been found to be preferentially localized to the dense subfractions of LDL, which are known to be highly capsular. Therefore, plasma Lp-PLA ₂ Levels readily measure these small, dense LDL particles, which are highly tunic. The presence of the enzyme in a patient's blood sample is detected by analysis of enzyme activity (ie, by an assay set up for the purified enzyme) or using polyclonal or monoclonal antibodies prepared for the purified enzyme. It is expected that it can be assayed by assaying the protein content of the sample. Monoclonal (and polyclonal) antibodies raised against the purified enzyme or fragments thereof are novel per se and form a further aspect of the invention. Data and Example 1. Lp-PLA ₂ Screening for Inhibition The turnover rate of the artificial substrate (A) in 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer containing 150 mM NaCl, pH 7.4 at 37 ° C was determined. The enzyme activity was quantified by measuring. Assays were performed in 96 well titer plates. Lp-PLA ₂ Were pre-incubated with vehicle or test compound in a total volume of 180 μl for 10 minutes at 37 ° C. The reaction was then initiated by adding 20 μl 10 × substrate (A) to give a final substrate concentration of 20 μM. The reaction was followed for 20 minutes at 405 nm using a plate reader capable of automatic mixing. The reaction rate was measured as the rate of change of absorbance. result: 2. Copper stimulated LDL oxidation Copper stimulated oxidation of LDL is routinely measured by monitoring the change in absorbance at 234 nm and following the increase in conjugated diene formation. This assay can be used to study inhibitors of oxidative modification of LDL. Fig. 1 shows the Lp-PLA due to the extension of the lag phase. ₂ It is shown that the inhibitor is an effective inhibitor of LDL oxidation using compound 4 as an example. 3. Cu ²⁺ Inhibition of stimulated lyso-phosphatidylcholine (lyso-PtdCho) formation A 1 ml aliquot of human LDL (0.25 mg protein / ml) was incubated with compound or vehicle for 15 minutes at 37 ° C. Then 5 μM Cu ²⁺ Was added to cause oxidation / lyso-PtdCho formation. The incubation was stopped by adding 3.75 ml chloroform / methanol / cHCl (200: 400: 5, v / v / v). After adding 1.25 ml chloroform and 1.25 ml 0.1M HCl, the mixture was vortexed and centrifuged. The lower layer was carefully removed and the upper layer was re-extracted with an equal volume of synthetic lower layer. The extracts were pooled and dried under nitrogen. Phospholipids were reconstituted in 50 μl chloroform / methanol (2: 1 v / v). A 10 μl aliquot was spotted on a pre-run silica gel HPTLC plate and then developed in chloroform / methanol 25-30% methylamine (60: 20: 5 v / v / v). The plate was then sprayed with the fluorescent indicator 2-p-toluidinylnaphthalene-6-sulfonic acid (50 mM Tris / HCl, 1 mM in pH 7.4) to identify the phospholipid component. Fluorescence was measured at 222 nm using a CAMAG TLC scanner. Lipoprotein lyso-PtdCho content was quantified using a standard curve prepared in parallel (0.05-0.5 μg). Compound 4 dose resulted in an accumulation of LDL lyso-PtdCho stimulated by copper ions of ˜30 μM IC. ₅₀ Inhibits value-dependently. 4. Lipoprotein related Lp-PLA ₂ Purification of low density lipoprotein (LDL) was obtained by plasmapheresis. LDL was dialyzed overnight at 4 ° C. against 0.5 M NaCl, 50 mM MES (4-morpholineethanesulfonic acid) (pH 6.0). Solid CHAPS (3-[(-3-colamidopropyl) dimethylamino] -1-propanesulfonate) was added to 10 mM and the LDL was solubilized by stirring for 30 minutes. Solubilized LDL was pumped onto a pre-equilibrated Blue Sepharose 6FF (Pharmacia) column (2.6 x 20 cm). The column was then loaded with 50 mM MES, 10 mM CHAPS, 0.5 M NaCl, pH = 6.0, then 50 mM Tris, 10 mM CHAPS, 0.5 M NaCl, pH = 8 until the absorbance of the eluate reached zero (280 nm). Washed with 0.0. Lp-PLA using 50 mM Tris, 10 mM CHAPS, 1.5 M NaCl, pH = 8.0 ₂ Was eluted. Then Lp-PLA ₂ Fractions were centrifuged and dialyzed overnight against 50 mM Tris, 10 mM CHAPS, pH = 8.0. Dialyzed Lp-PLA ₂ Was subjected to anion exchange chromatography on a Mono Q column (Phar macia) using 50 mM Tris, 10 mM CHAPS, pH = 8.0 with a 0-0.3 M NaCl gradient. Lp-PLA obtained from Mono Q column ₂ Fractions were applied directly to a Hi Trap Blue cartridge (Pharmacia). The cartridge was washed with 50 mM Tris, 10 mM C HAPS, 1.5 M NaCl, pH = 8.0 until the absorbance of the eluate (280 nm) became zero. Then, Lp-PLA was prepared using 50 mM Tris, 10 mM CHAPS, 1.5M NaCl, and pH8. ₂ Was eluted. From now on Lp-PLA ₂ (This is over 95% pure as shown in FIG. 2). This also indicates that the natural enzyme is extensively glycosylated. 5. Enzyme Sequence The purity of the final enzyme preparation was confirmed by the following 5 criteria: 1) SDS-polyacrylamide gel electrophoresis gives one band for both native and deglycosylated forms 2) Reversed phase fast Liquid Liquid Chromatography (RP-HPLC) gives a single peak, 3) no protein sequencing results from the pristine preparation, which indicates that the protein is blocked at the N-terminus. , And implied that there were no contaminants with open N-termini), 4) laser desorption mass spectrometry observed only one broad peak for the deglycosylated protein, and 5) None of the sections of extended peptide data from sequencing provided any database code to indicate contaminating proteins. Internal sequence information was obtained using three cleavage methods [trypsin (after deglycosylation), cyanogen bromide (methionine cleavage) and BNPS-Skatol (tryptophan cleavage)]. The resulting peptides were separated by RP-HPLC, pooled and sequenced. From accumulated sequence data, Lp-PLA ₂ Some extension of the primary structure of the enzyme was confirmed. These are shown below as peptides 1, 2, 3 and 4 (SEQ ID NOs: 1-4). When searched against the National Center for Biotechnology Information (NCBI) non-redundant peptide sequence database, no high similarity was obtained. Estimation of the molecular weight of pure deglycosylated protein by laser desorption mass spectrometry gave values in the region of 45-47 kDa (separated into 45 kDa and 46-47 kDa). This indicates that the sequence makes up about 15-20% of the protein. 6. Gene Sequences Three expressed sequence tags (ESTs) from a human cDNA library were found to have extensive consistency with peptide sequences 1-3. These ESTs are shown below as nucleotide sequences 1-3 (SEQ ID NOs: 5-7). Nucleotide sequence 1 is a 420 base sequence derived from a human fetal spleen library. Nucleotide sequence 2 is a 379 base sequence from a 12-week human embryo library. Nucleotide sequence 3 is a 279-base sequence derived from the T cell lymphoma library. The identities at both the nucleic acid and amino acid levels are identical for all three ESTs [nucleotide sequence 3 (bases 1-278), 1 (bases 1-389) (in reverse orientation) and 2 (bases 1-304)]. It justifies the overlapping consistency of the cDNA with the peptide sequences 1, 2 and 3 (partial). Above these ranges, the resolution of unprocessed sequence data is low, and accurate base calling is impossible. There are two unassigned peptide segments remaining from peptides 3 and 4, both of which are expected to be present in the complete protein, -QYINPAV- and WLMGNILRLL-FGSMTTPAN-. 7. Full length Lp-PLA ₂ Isolation of cDNA The total DNA sequence of the clone (HLTA145) giving the lymphoma EST (SEQ ID NO: 7) was determined to obtain all 572 bases; SEQ ID NO: 8. There is one base difference between this sequence and the EST (between bases 1-256 of the EST). That is, there is an A at position 27 of HL TA145, but there is a T at the EST. This changes the decryption to be L in HLTA 145 versus F in the EST. Full length Lp-PLA ₂ To isolate clones, the lymphoma cDNA library was screened using clone HLT A145 as a radiolabeled probe. The library was prepared in the bacteriophage lambda vector, Unizap XR (Stratagene). Preparation of filters for screening The library was plated on E. coli XL-1 blue host cells at a density of 20,000 plaques per 150 mm Petri dish (ie 200,000 plaques on 10 dishes). 100 ml LB / 0.2% w / v maltose / 10 mM MgSO _Four Medium XL-1 blue was prepared overnight. The cells are pelleted and 50 ml of 10 mM MgSO 4. _Four Resuspended in and stored on ice. 180 μl of library bacteriophage stock (23,400 pfu) was added to 7 ml of XL-1 blue cells, mixed and divided into 10 aliquots of 615 μl. Ten tubes were incubated for 30 minutes at 37 ° C. Add 7 ml of melted (45 ° C) top agarose (0.7% w / v agarose in LB), mix well and onto a 150 mm LB agar plate (1.5% w / v agar in LB). I poured it. The plate was turned over and incubated at 37 ° C. for about 7.5 hours. The plate was kept at 4 ° C until needed. The plaques were transferred to the filter by overlaying a 132 mm Hybond-N nylon filter (Amersham International) on the plate for 2 minutes (4 minutes in duplicate). The DNA on the filter was denatured for 2 minutes (0.5M NaCl, 1.5M NaOH) and neutralized for 4 minutes (1.5M NaCl, 1.0M Tris pH 7.4) and the filter was placed on 2 × SSC. I put it on for 1 minute. The filter is then dried to 120,000 μJoules / cm. ² The DNA was cross-linked to the filter using Stratalinker-UV2400 (Stratagene). 3 hours at 55 ° C, 1 mM EDTA, 0.5M NaHPO in Techne HB2 hybridization oven. _Four The filters were prehybridized in 7% SDS (Church, GM and Gilbert, W. (1984) PNAS USA 81, p. 1991-1995). Each bottle contained two filters and 25 ml of prehybridization solution. Preparation of radiolabeled probe The probe cDNA (from HLTA145) was excised as an approximately 600 bp EcoRI-Xho I fragment from pBluescript II SK +/-, TaqDNA polymerase (Boehringer Mannheim) and EST. 1.2 MBq by PCR labeling with primers designed to prime at the 5'and 3'ends of the sequence ³² PdATP and 1.2MBq ³² About 100 ng of the gel purified fragment was labeled with P d CTP. The labeling reaction was carried out in a total volume of 200 μl, which contained the following concentrations of unlabeled dNTPs. dATP 20 μM dCTP 20 μM dGTP 200 μM dTTP 200 μM The PCR reaction was carried out for 35 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds. Screening Radiolabeled probe was denatured at 98 ° C for 5 minutes and divided into 10 aliquots of 20 μl. One aliquot was added per hybridization bottle. Hybridization was carried out at 55 ° C. for 16 hours. The filter was washed at 60 ° C. (2 × 10 minutes) with 0.1% w / v SDS, 0.1 × SSC (50 ml / wash / bottle). The filter was subjected to autoradiography, and after exposure for 5 days, the film (Fuji Medical X-Ray Film) was developed. Double positives were advanced to the secondary screen. The plaque was treated with 1 ml SM (100 mM NaCl, 10 mM MgSO 4 _Four 1 M Tris, pH 7.4), titrated and plated on 90 mm Petri dishes at 20-200 pfu / dish. Secondary screening was performed in the same manner as the primary screening except that the filter was washed at 65 ° C. After 16 hours of exposure, the autoradiograph was developed. DNA Sequencing Double positive clones from the secondary screen were excised and characterized from the λ Unizap XR bacteriophage vector into the Bluescript phagemid (by the Stratagene manual). One of the clones, which has an insert of approximately 1.5 kb, was subjected to primer walking on both strands (USB Sequenase 2.0 DNA Sequencing Kit ( se queencing kit)) The sequence was determined (SEQ ID NO: 9). The cDNA has a potential open reading frame that encodes a 441 amino acid polypeptide. The 3'region of the full-length cDNA matches the HLTA145 sequence except for three mismatches (see below). Lymphoma Lp-PLA ₂ The predicted polypeptide sequence of is shown as SEQ ID NO: 9. A close examination of the full length cDNA (SEQ ID NO: 9) reveals a possible error in Peptide 3. One of these mistakes lies in the assignment of continuity between VMs that conflicts with perfect sequence pairing with the cDNA after this position. A short peptide containing the sequence -QYI -NP- was purified with a longer cyanogen bromide partially cleaved peptide and was assigned as the main sequence by virtue of its abundance and appeared to be adjacent to the next amino acid. The remaining segment of peptide 3 and all of peptide 4 can be identified in the predicted full-length enzyme sequence (SEQ ID NO: 9). Thus, Peptide 3 is actually two separate peptides 5 (SEQ ID NO: 10) and 6 (SEQ ID NO: 11). A second possible error has occurred in the transcription from the raw data for peptide 3, which upon confirmation was consistent with peptide 5 having the sequence -QYINPVA rather than the sequence -QY -INPAV-. The differences between the three bases are as follows. 1) T at position 859 is A in HLTA145; F in full-length amino acid change, and L in HLTA145 (note that the original EST is consistent with full-length cDNA at position 859). 2) C at position 1173 is T in HLTA145; A in the entire amino acid change and V in HLTA145. 3) T at position 1203 is C in HLTA145; L in the entire amino acid change, and S in HLTA145. The peptide theta and fetal splenic EST sequences (SEQ ID NO: 5) support the full length cDNA sequence for differences (2) and (3), while human embryonic EST (SEQ ID NO: 6) is at position 1173 at the lymphoma EST (SEQ ID NO: 6). It matches with 7). Furthermore, human embryo EST (SEQ ID NO: 6) has a difference (4) corresponding to position 1245 in the full length lymphoma sequence (SEQ ID NO: 9) (bases 2-304 of human embryo EST and full length lymphoma cDNA). Comparison between). 4) A at position 1245 is T in embryonic EST (SEQ ID NO: 6) [amino acid change from D to V (in embryonic EST)]. Peptide data containing this region supports the lymphoma DNA sequence (SEQ ID NO: 9). Lp-PLA of 848 to 1361 of SEQ ID NO: 9 (amino acid residues 271 to 441 of SEQ ID NO: 9) ₂ The DNA sequence is the region in which all major data (ie peptide data, fetal spleen, full-length lymphoma) are made to substantially match, and it contains the known active site, and thus Lp-PLA. ₂ It is considered to be an important characteristic region of the enzyme. The estimated molecular weight for all reading frames is 50090. This exceeds that determined for deglycosylated, purified proteins, but post-translational events explain this discrepancy. Most likely of these are removal of the N-terminal signal peptide and / or C-terminal restricted proteolysis. The latter will occur in vivo during purification or under deglycosylation conditions. Diagnostic Method Blood samples were taken from patients, plasma / serum samples were prepared and passed through a dextran sulfate column pre-equilibrated with 0.9% (w / v) NaCl solution. After washing with the same salt solution, Lp-PLA with 4.1% (w / v) NaCl solution ₂ Was eluted. Also, heparin agarose columns can be used with wash and eluent containing 0.7% and 2.9% NaCl respectively. The enzyme present in the sample was (p) Lp-PLA by monitoring the change in absorbance at 400 nm using the enzyme activity [substrate (A) (see structure 1 in 1)]. ₂ Assay activity. The purified enzyme is pre-incubated at 37 ° C. and after 5 minutes substrate (50 μM) is added. The change in absorbance at 400 nm is monitored for 20 minutes. This substrate is already a classical calcium-dependent PLA ₂ (Washburn, WN and Dennis, EA, Journal of American Chemical Society (J. Amer Chem. Soc.), 1990. , 112, 2040-2041)]; or (b) protein content [total protein content (ie, enzyme content) is that of purified human Lp-PLA]. ₂ Can be quantified using the polyclonal antisera raised against. The antiserum recognizes both native and glycosylated enzymes as determined by immunoprecipitation of activity and Western blot analysis]. Polyclonal antiserum was prepared as follows. For immunization of rabbits, 0. 5 ml of purified human Lp-PLA ₂ It was included to mix (= 100 μg) with an equal volume of complete Freund's adjuvant. The final emulsion was administered subcutaneously as a 4 × 0.25 ml injection. A booster immunization with incomplete Freund's adjuvant / antigen mixture (4 × 0.25 ml sc; dose = 50 μg) was given 4 weeks later. Appropriate titers were clearly seen between 6 and 8 weeks after the first injection. In the drawings FIG. 1 is a graph of absorbance at 234 nm versus time (min) in a study of inhibition of copper (5 μM) -stimulated LDL (150 μg / ml) oxidation by Compound 4 versus control vehicle. FIG. 2 shows purified Lp-PLA of Example 4 after separation by polyacrylamide gel electrophoresis. ₂ It is an analysis of materials. Lanes 2, 4 and 6 are purified native Lp-PL A. ₂ Containing adjacent fractions of. Lanes 1, 3 and 5 are fractions 2, 4 and 6 after N-deglycosylation, respectively. Sequence data SEQ ID NO: 1-Peptide 1 SEQ ID NO: 2-Peptide 2 SEQ ID NO: 3-Peptide 3 SEQ ID NO: 4-Peptide 4 SEQ ID NO: 5-nucleotide sequence 1 SEQ ID NO: 6-nucleotide sequence 2 SEQ ID NO: 7-nucleotide sequence 3 SEQ ID NO: 8-DNA sequence of HLTA145 SEQ ID NO: 9-Lymphoma Lp-PLA ₂ CDNA sequence SEQ ID NO: 10-Peptide 5 SEQ ID NO: 11-peptide 6

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI C07K 16/40 8318-4H C12N 15/09 ZNA C12P 21/08 9358-4B C12Q 1/44 6807-4B G01N 33 / 53 D 8310-2J 33/573 A 8310-2J // A61K 39/395 D 9284-4C P 9284-4C (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), JP, US (72) Inventor Tew, David Graham Hearts Aell 6.9 Aal, Welwin, The Frith Street address not displayed) SmithKline Beatham Pharmaceuticals (72) Inventor Southern, Christopher Donald I Squirrel Country Hertz Eyle 6.9 Aal, Welwyn, The Frith (No street numbering) SmithKline Beatham Pharmaceuticals (72) Inventor Hicky, Diadori Mary Bernadette United Kingdom Hearts Ahl 6.9 Aal, Welwyn, The Frith (No street address) SmithKline Beatham Pharmaceuticals (72) Inventor Grosier, Israel Simon Essex C19.5 Eddie, England , Harlow, The Pinnacles, Cold Harbor Road (no street numbering) SmithKline Beecham Pharmaceuticals (72) Inventor Lawrence, Jeffrey Mark Prouse Essex CM 19 / 5E, England Idy, Harlow, The Pinnacle , Cold Harbor Road (no street number) SmithKline Beecham Pharmaceuticals (72) Inventor Rice, Simon Quentin John Essex CM 19.5 Aid, Harlow, The Pinnacles , Cold Harbor Road (no street address) SmithKline Beecham Pharmaceuticals

Claims (1)

[Claims] 1. Enzyme in purified form Lp-PLA ₂ . 2. Enzyme Lp-PLA according to claim 1, characterized by one or more partial peptide sequences selected from SEQ ID NO: 1, 2, 3, 4, 10 and 11 and / or by having a molecular weight of at least 45 kDa. ₂ . The enzyme Lp-PLA _{2 according} to claim 1 or 2, which has a molecular weight of 3.45 kDa. The enzyme Lp-PL A _{2 according} to claim 1 or 2, having a molecular weight of 4.45 to 50 kDa. The enzyme Lp-PLA _{2 according to} claim 4, which has a molecular weight of 5.45 to 47 kDa. The enzyme Lp-PLA _{2 according to} claim 5, having a molecular weight of 6.46-47 kDa. 7. The enzyme Lp-PLA _{2 according} to claim 1, characterized by a partial peptide sequence corresponding to residues 271-441 of SEQ ID NO: 9. 8. The enzyme Lp-PLA _{2 according} to claim 1 having the sequence given by SEQ ID NO: 9 or an enzyme having Lp-PLA ₂ activity and being substantially homologous to SEQ ID NO: 9 or a fragment thereof. 9. An enzyme fragment selected from SEQ ID NOs: 1, 2, 3, 4, 10 and 11. 10. An isolated nucleic acid molecule encoding Lp-PLA ₂ or an antisense analog thereof. 11. An isolated nucleic acid molecule encoding the enzyme or fragment of any one of claims 1-9 or an antisense analog thereof. 12. The isolated nucleic acid molecule according to claim 10, which comprises the bases 1 to 389 of SEQ ID NO: 5; the bases 1 to 304 of SEQ ID NO: 6; the bases 1 to 278 of SEQ ID NO: 7; or the sequence corresponding to SEQ ID NO: 8. Antisense analog. 13. The isolated nucleic acid molecule according to claim 11, which comprises a sequence corresponding to bases 848 to 1361 of SEQ ID NO: 9, or an antisense analog thereof. 14. An isolated nucleic acid molecule according to claim 10 consisting of bases 1-1361 or 38-1361 of SEQ ID NO: 9 or encoding an enzyme having Lp-PLA ₂ activity and being substantially homologous to said isolated molecule. Is a nucleic acid molecule, or an antisense analog thereof. 15. A recombinant vector comprising the nucleic acid molecule according to any one of claims 10 to 14. 16. A host cell comprising the molecule according to any one of claims 10 to 14. 17． Use in the treatment of inhibitors of the enzyme Lp-PLA ₂ . 18. Use of inhibitors of Lp-PLA ₂ in atherosclerosis. 19. A method of diagnosing a patient's susceptibility to atherosclerosis, which comprises taking a blood sample from a patient and analyzing the sample for the presence of the enzyme Lp-PLA ₂ . 20. 21. The method of claim 19, wherein analyzing the sample comprises assaying the sample for enzyme activity. 21. 20. The method of claim 19, wherein analyzing the sample comprises assaying the sample for protein content using polyclonal or monoclonal antibodies raised against the enzyme. 22. A polyclonal antibody raised against the purified Lp-PLA ₂ enzyme of any one of claims 1-8. 23. A monoclonal antibody raised against the purified Lp-PLA ₂ enzyme of any one of claims 1-8. 24. Inhibiting an isolated enzyme Lp-PLA ₂ by contacting it with a test compound and measuring the turnover rate of the enzyme substrate compared to the turnover rate in the absence of the test compound A method of screening a compound to identify the compound.